Pituitary tumors are the second most common adult primary brain tumor, with a variable clinical course. Recent work has identified a number of genetic determinants of pituitary tumor subtypes, which may augment traditional histopathologic classification schemes. We sought to determine whether pituitary tumors could be stratified based on objective molecular characteristics using a clinical genomics assay. We performed a retrospective analysis of patients operated on at the Brigham and Women's Hospital from 2012 to 2016 whose pituitary tumors were profiled using multiplexed next-generation sequencing. We analyzed 127 pituitary tumors, including 114 adenomas, 5 craniopharyngiomas, and 8 tumors of other histologies. We observed recurrent BRAF^V600E mutations in papillary craniopharyngiomas, CTNNB1 mutations in adamantinomatous craniopharyngiomas, and activating GNAS mutations in growth hormone–secreting adenomas. Furthermore, we validated the presence of two distinct genomic subclasses in adenomas (i.e., those with disrupted or quiet copy-number profiles) and the significant association of disruption with functional hormone status (P < 0.05). We report the clinical implementation of next-generation sequencing of pituitary tumors. We confirmed previously identified molecular subclasses for these tumors and show that routine screening as part of clinical practice is both feasible and informative. This large-scale proof-of-principle study may help to guide future institutional efforts for pituitary tumor classification as well as the incorporation of such techniques into prospective analysis as part of clinical trials.